PROJECT SUMMARY/ABSTRACT The epidemiology of the hepatocellular carcinoma (HCC), the most common form of liver cancer, is shifting from being associated with viral-related to non-alcoholic steatohepatitis (NASH)-related HCC. Thus, NASH-HCC is currently the biggest public health problem for this type of liver cancer. While obesity and diabetes are two of the factors associated with NASH, it also develops in lean individuals that have diet-related visceral obesity (i.e., fat deposits within the visceral cavity). While the molecular basis of NASH-HCC in lean/viscerally obese individuals remains undefined, the dietary molecule choline may play an important role in its development. In the context of non-alcoholic fatty liver disease (NAFLD), choline can lower the risk of NAFLD in normal weight women when present in the diet or lead to NAFLD development in animal models when absent from the diet. Gene methylation by choline in the diet can affect carcinogenic signaling pathways and consequently cancer development. Other dietary molecules such as fat, fructose, and cholesterol also have an impact on NAFLD through liver inflammation and progression to NASH. In addition, lipid metabolism is altered during NASH-HCC progression, including altered levels of fatty acids and ceramides. The goal of the proposed research is to understand the mechanisms of lean-associated NASH-HCC compared to obesity-associated NASH-HCC. This will be accomplished through two specific aims: 1) identify the fatty acids and ceramides that are altered in mice fed a Western diet deficient in choline that leads to lean NASH-HCC and 2) identify molecular pathways that are differentially expressed in the development of lean versus obese NASH-HCC in animals fed a Western diet that is deficient or supplemented in choline. NASH and HCC will be induced using a Western diet high in trans-fat, fructose, and cholesterol (HFFC). Using mass spectrometry of plasma samples, unique lipid metabolites in lean mice with HCC compared to obese mice with HCC will be identified, and mRNA sequencing will be carried out in tumors from: 1) normal weight mice fed an HFFC and choline deficient diet and 2) obese mice fed an HFFC and choline supplemented diet. Unique metabolite patterns and differentially expressed genes will be identified by comparing the two groups. Based on studies in other mouse models and on studies involving humans with HCCs, the hypothesis is that lean NASH-HCC mice will have different levels of proteins associated with de novo fatty-acid synthesis, inflammation, and fibrogenesis compared to obese NASH-HCC mice. The identification of molecular mechanisms associated with lean NASH-HCC in this project will provide information on how dietary components such as choline can affect carcinogenic pathways and potentially prevention of metabolic-based liver cancer. Thus, this project fits well within the Nebraska Center for Prevention of Obesity through Dietary Molecules (NPOD)'s focus of identifying biological food-borne signals that prevent, treat, and cure obesity and obesity- related diseases and will lay important ground work for Dr. Farazi's long-term goal to develop systems for prevention and early detection of liver cancer.